摘要
目的探究微小RNA(miR)-124靶向调控趋化素样因子超家族成员6(CMTM6)对胶质瘤细胞的侵袭、迁移的影响。方法实时荧光定量PCR(qPCR)法检测OLN93、SHG-44、U87、U251、A172细胞中miR-124、CMTM6 mRNA表达水平;蛋白免疫印迹实验检测OLN93、SHG-44、U87、U251、A172细胞中CMTM6蛋白表达水平。miRTarBase预测miR-124与CMTM6的结合位点,实验设mimic NC组、miR-124 mimic组、miR-124 mimic+pcDNA组、miR-124 mimic+CMTM6组,分别转染miR-124 mimic NC、miR-124 mimic、pcDNA、pcDNA+CMTM6于各组细胞中。qPCR法检测各组细胞中miR-124、CMTM6 mRNA表达水平;CCK-8检测细胞增殖情况;Transwell检测细胞侵袭、迁移情况;划痕实验检测细胞迁移情况;蛋白免疫印迹实验检测细胞中CMTM6、细胞表面程序性死亡蛋白配体1(PDL1)蛋白表达水平。结果与OLN93相比,SHG-44、U87、U251、A172细胞中miR-124表达水平降低(P<0.05),CMTM6 mRNA和蛋白表达水平升高(P<0.05),选择U251细胞进行后续实验。miRTarBase预测发现miR-124与CMTM6存在结合位点,并经双荧光素酶报告基因实验验证。与mimic NC组和miR-124 mimic+CMTM6组相比,miR-124 mimic组和miR-124 mimic+pcDNA组细胞中miR-124表达水平升高(P<0.05),CMTM6 mRNA表达水平降低。培养24、48、72、96 h时细胞OD450降低,侵袭、迁移细胞数量减少,CMTM6、PD-L1蛋白表达水平降低(P<0.05)。结论上调miR-124表达可靶向下调CMTM6的表达,从而抑制胶质瘤细胞的侵袭、迁移。
Objective To investigate the inhibitory effect of microRNA(miR)-124 targetingly regulating chemokinelike factor super family 6(CMTM6)on the invasion and migration of glioma cells.Methods The levels of miR-124 and CMTM6 mRNA in OLN93,SHG-44,U87,U251 and A172 cells were detected by real-time fluorescence quantitative PCR(qPCR).The levels of CMTM6 protein in OLN93,SHG-44,U87,U251 and A172 cells were detected by Western blot assay.The miRTarBase was used to predict the binding sites of miR-124 and CMTM6.There were four experimental groups in this study:mimic NC group,miR-124 mimic group,miR-124 mimic+pcDNA group and miR-124 mimic+CMTM6 group.The mimic NC,mimic miR-124,pcDNA and pcDNA+CMTM6 were transfected into each cell group.The levels of miR-124 and CMTM6 mRNA were detected by qPCR.CCK-8 was used to detect the cell proliferation.Transwell test was used to detect cell invasion and migration.The cell migration was detected by scratch test.The protein levels of CMTM6 and cell surface programmed death protein ligand 1(PD-L1)were detected by Western blot assay.Results Compared with OLN93 group,the levels of miR-124 were lower in SHG-44,U87,U251 and A172 groups(P<0.05).The levels of CMTM6 mRNA and protein were higher(P<0.05).U251 cells were selected for subsequent experiments.miRTarBase predicted that there was a binding site between miR-124 and CMTM6,which was verified by double luciferase reporter gene experiment.Compared with that in mimic NC group and miR-124 mimic+CMTM6 group,the levels of miR-124 in miR-124 mimic group and miR-124 mimic+pcDNA group were higher(P<0.05),and the level of CMTM6 mRNA was lower.After cells were cultured for 24,48,72,96 h,the cell OD450 decreased,the numbers of invasion and migration cells decreased,the protein levels of CMTM6 and PD-L1 were lower(P<0.05).Conclusion Increasing the expression of miR-124 can down-regulate the expression of CMTM6,thereby inhibiting the invasion and migration of glioma cells.
作者
梁博
王新军
王建业
周少龙
杨卓
LIANG Bo;WANG Xin-jun;WANG Jian-ye;ZHOU Shao-long;YANG Zhuo(Department of Neurosurgery,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《天津医药》
CAS
北大核心
2021年第3期225-230,I0001,共7页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(81972361)。
